The present invention relates to vaccines and methods of vaccination which involve transfection of cells.
Vaccination has been an important medical pursuit ever since it was observed that, for certain diseases, initial exposure to the infectious agent conferred lifelong immunity against subsequent infections. Vaccines have been used for many years in order to build immunity in an individual against infection by particular pathogenic organisms such as viruses, bacteria, fungi and parasites.
Early vaccines relied on live organisms or killed organisms that retained their immunogenicity. A better understanding of the structure and function of particular pathogens and of the mechanisms of adaptive immunity has made it possible to design safer and more directed vaccines. The current vaccine against Hepatitis B virus, for example, relies on inoculation using only a portion of the viral surface antigen, rather than the complete organism. Such directed vaccines lead to fewer side-effects and avoid unwanted immune responses to antigens that are not protective, i.e., do not confer lasting immunity.
Advanced vaccine design has made use of recombinant DNA technology and gene therapy concepts to provide DNA vaccines, wherein a vector for expression of a desired immunogen in mammalian cells is injected into a subject. The vector is taken up into cells around the site of injection, and then operates to express the immunogen in situ, which in turn leads to a protective immune response.
A problem associated with the use of DNA vaccines thus far is that the tissue into which the vector is typically injected, i.e., muscle, is not usually associated with antigen presentation. Consequently, the DNA vaccine does not produce a highly effective immune response.
There is a need for methods and specialized gene delivery vehicles suitable for delivery of complex antigens to cells so that the resulting antigen presentation is capable of activating substantially all of the components of the adaptive immune system, i.e., capable of eliciting the immune response necessary to combat a particular pathogen whether mediated by antibodies, cytotoxic T cells, helper T cells, natural killer cells, or macrophages.
The present invention provides methods and compositions for obtaining long-lasting immunity via delivery to an antigen presenting cell a complex comprising a nucleic acid encoding a first epitope, and a peptide containing a second epitope.
Antigen presenting cells present antigenic epitopes of antigens to T cells in association with either Class I or Class II MHC molecules. Endogenously synthesized proteins are generally presented in association with Class I whereas proteins taken up from the environment (exogenously synthesized proteins) are generally presented in association with Class II. It is therefore beneficial for a vaccine to produce presentation of the required antigen to both Class I and II MHC molecules. The invention provides for stimulation of both class I and II presentation by providing antigen to an antigen presenting cell in the form of an endogenously synthesized protein (i.e., a nucleic acid-encoded epitope) and an exogenously synthesized protein.
It is believed that the dual delivery to an antigen presenting cell of an epitope, or antigen, in its peptide or polypeptide form, together with a second nucleic acid encoded epitope results in an enhanced immune response.
The invention therefore encompasses a method of vaccinating a mammal against a disease, comprising administering to said mammal a mixture of (i) a nucleic acid encoding a first epitope and (ii) a peptide comprising a second epitope such that the nucleic acid and the peptide are taken up by and the nucleic acid is expressed in a professional antigen presenting cell of the mammal, wherein an immune response is elicited in the mammal to the epitopes.
The invention also encompasses a method of vaccinating a mammal against a disease wherein the mixture administered comprises a complex comprising (i) a nucleic acid encoding a first epitope and (ii) a peptide comprising a second epitope such that the complex is taken up by and expressed in a professional antigen presenting cell of the mammal, wherein an immune response is elicited in the mammal to the first and second epitopes.
The invention also encompasses a method of vaccinating a mammal against a disease, comprising administering, to the mammal professional antigen presenting cells containing (i) a recombinant nucleic acid encoding a first epitope and (ii) a second epitope that is not normally present in the antigen presenting cells and, wherein upon administration an immune response is elicited in the mammal to the epitopes.
The invention also encompasses a composition for vaccinating a mammal against a disease, comprising a complex comprising (i) a vector comprising a nucleic acid encoding a first epitope and a sequence which permits maintenance of the vector in episomal form , and (ii) a peptide containing a second epitope, wherein said composition is adapted for delivery to or selective expression in antigen presenting cells.
The invention also encompasses a complex for vaccinating a mammal against a disease, comprising professional antigen presenting cells containing (i) a recombinant nucleic acid encoding a first epitope and (ii) a peptide containing a second epitope that is not normally present in the antigen presenting cells.
The invention encompasses a method of vaccinating a mammal against a disease, comprising administering to the mammal a complex comprising (i) a nucleic acid and (ii) a peptide including an epitope such that the complex is taken up by a professional antigen presenting cell of the mammal, wherein an immune response is elicited in the mammal to the epitope.
In this aspect of the invention, it is believed that the presence of the nucleic acid in the mixture promotes uptake by antigen presenting cells of the peptide containing an epitope so as to promote an immune response to the epitope.
Preferably, the first epitope is from an infectious agent or an organism, and the first epitope is present in the mammal during the course of a disease.
It is preferred that the complex further comprises a second epitope, and that the first and second epitopes are epitopes of the same antigen, or epitopes of the same infectious agent or organism.
For example, the first and/or second epitope may comprise an immunodominant epitope of influenza NP.
It is also preferred that the complex or mixture further comprises a second peptide that contains an epitope that is different from the epitope contained in the peptide referred to in (ii) above.
The complex may further comprise a cell-targeting ligand for targeting professional antigen presenting cells.
It is preferred that the nucleic acid is intimately associated with the peptide containing the epitope such that the nucleic acid is preferably in condensed form.
Therefore, it also is preferred that the first epitope is present in a two-domain polypeptide comprising the first epitope fused to a nucleic acid-binding amino acid sequence. As used herein, the term xe2x80x9cepitopexe2x80x9d refers to an immunogenic amino acid sequence. An epitope may refer to a minimum amino acid sequence of 6-8 amino acids (i. e., a peptide), which minimum sequence is immunogenic when removed from its natural context and is carried in a complex according to the invention as a peptide, or when transplanted into a heterologous polypeptide such that it retains its natural immunogenicity and thus is carried in a complex according to the invention as part of a polypeptide. An epitope also may refer to that portion of a natural polypeptide which is immunogenic, where the natural polypeptide containing the epitope is referred to as an antigen. Of course, a polypeptide or antigen may contain one or more distinct epitopes. An epitope also may refer to an immunogenic portion of a multichain polypeptide, i.e., which is encoded by distinct open reading frames. The terms epitope, peptide, and polypeptide all refer to a series of amino acids connected one to the other by peptide bonds between the alpha-amino and alpha-carboxy groups of adjacent amino acids, and may contain or be free of modifications such as glycosylation, side chain oxidation, or phosphorylation, provided such modifications, or lack thereof, do not destroy immunogenicity. As used herein, the term xe2x80x9cpeptidexe2x80x9d is meant to refer to both a peptide and a polypeptide or protein.
It is desirable that the epitope (peptide, polypeptide, antigen) be as small as possible while still maintaining immunogenicity. Immunogenicity is indicated by the ability to elicit an immune response, as described herein, for example, by the ability to bind an appropriate MHC molecule (i.e., an MHC class I or class II molecule) and to induce a T cell response and an antibody response, e.g., by measuring a cytotoxic T cell response or a serum antibody response to a given epitope(s).
As used herein, the terms xe2x80x9cantigenxe2x80x9d or xe2x80x9cimmunogenxe2x80x9d refer to a peptide, protein, polypeptide which is immunogenic, that is capable of eliciting an immune response in a mammal, and therefore contains at least one and may contain multiple epitopes. According to the invention a xe2x80x9cpathogenxe2x80x9d, organism, or xe2x80x9cagentxe2x80x9d may cause a disease for which vaccination is desired according to the invention. As used herein, these terms refer to a virus, bacteria, fungus, or a parasite. The term xe2x80x9cagentxe2x80x9d also may refer to antigens such as tumor antigens or antigens associated with auto-immunity. The invention does not contemplate administration of a whole pathogen, or its entire genome, to achieve antigenicity. Therefore, the term xe2x80x9cepitopexe2x80x9d is limited to this extent that it does not refer to a whole pathogen.
xe2x80x9cImmune responsexe2x80x9d refers to either a cellular or a humoral immune response or to both a cellular and a humoral immune response.
It is believed that the antigenic peptide or protein delivered to the target cell by the delivery vehicle will be processed as an exogenously synthesized antigen and epitopes will be presented predominantly in association with Class II MHC molecules to Class II-restricted T cells. The antigen encoded by the nucleic acid of the delivery vehicle will be endogenously synthesized and epitopes of the antigen will be presented predominantly in association with Class I MHC molecules. Presentation in association with both MHC Class I and II molecules will ensure a strong immune response.
It is preferred according to the invention that the nucleic acid encoded antigen is expressed only in professional antigen-presenting cells (APC).
As used herein, the term xe2x80x9cprofessional antigen-presenting cellsxe2x80x9d refers to MHC class II-bearing cells (that is, cells which express MHC class II or can be induced to express class II); such cells include B-lymphoctyes, dendritic cells other than follicular dendritic cells, macrophages, endothelial cells, phagocytic leukocytes, monocytes or monocyte derived cells, Kupffer cells, Langerhans cells, or stem cells thereof or other precursor cells, and other cells which either express class II or can be induced to express MHC class II.
The restricted expression of the gene to APCs or related cells can be achieved in two ways: 1) by targeting the delivery vehicle only to APCs or related cells and/or 2) by restricting the expression of the gene encoding the antigen to APCs or related cells.
It is contemplated according to the invention that the complex may further include a targeting ligand for targeting a receptor on the cell surface. Targeted delivery may be accomplished nonspecifically to both non-APCs and APCs, for example, where expression of the gene is restricted to APCs, or specifically to APCs only.
Thus, it is preferred that the complex of the present invention is adapted to target specifically to a professional antigen presenting cell of a mammal and/or the nucleic acid is adapted to be expressed specifically in a professional antigen presenting cell.
The term xe2x80x9cto target specifically to a professional antigen presenting cellxe2x80x9d refers to the situation where the complex is targeted to APCs and thus delivery will be substantially restricted to APCs. However, one skilled in the art will realize that targeting is rarely completely efficient and some delivery will occur to non-targeted cells. The term refers to where delivery to APCs is increased relative to other cell types, preferably by approximately two-fold or more.
Targeted delivery to APCs, their stem cells or other precursor cell types can be achieved by receptor-mediated gene transfer using delivery vehicles comprising the following examples of targeting ligands: (a) for hemopoietic stem cells: anti-CD34 monoclonal antibody, or the Stem cell factor (c-Kit or CD117), or flk-2 ligand (human homolog STK-1); (b) for monocyte/macrophage/dendritic cell precursors: anti-CD33 monoclonal antibody; (c) for differentiated macrophage/dendritic cells: glycosylated DNA binding peptides carrying mannose groups may be used to target to specific receptors, for example the mannose receptor; and (d) for MHC class II bearing cells: an antibody that is specific for the constant region of MHC class II proteins or a ligand that binds MHC class II, for example soluble CD4; for example, one subset of MHC class bearing cells, B lymphocytes, may be targeted using soluble CD4 or using antibodies to or ligands for CD80, CD19, or CD22; for endothelial cells, xcex3-interferon and the vascular endothelial growth factor (VEGF) receptors; and (e) for APCs or T cells ligands for or antibodies to co-stimulatory molecules such as B7-1, B7-2 or CD28, CTLA-4, respectively. These targeting ligands may play a dual role which involves increasing co-stimulatory signals to the APC; and thus increasing its activation, in addition to their targeting function.
Alternatively or in addition to the use of targeted complexes, DNA regulatory elements which lead to expression in APCs, their stem cells or other precursor cell types can be utilized.
The term xe2x80x9cto be expressed specifically in a professional antigen presenting cellxe2x80x9d refers to the situation where the expression of the nucleic acid is substantially restricted to APCs. The term therefore covers the situation where expression of the nucleic acid is restricted predominantly to APCs, for example, where approximately 50% and preferably 80%-100% of the cells in which expression of the nucleic acid occurs are APCs.
It is further contemplated according to the invention that the nucleic acid encoded antigen is predominantly expressed only in professional antigen-presenting cells (APCs).
Preferred regulatory elements include locus control regions (LCRs) such as the MHC class II LCR.
The present invention may be used to cause presentation of any protein or peptide capable of eliciting an immune response.
The present invention is applicable to any infectious agent against which an immune response can be measured. The invention also is applicable to other disease targets against which an immune response would be beneficial, such as tumors. Thus, the invention contemplates the use of a sequence encoding epitope of any infectious agent or disease target.
Preferably, the epitope is a key epitope, i.e., either the complete polypeptide sequence or an epitope which gives rise to a strong immune response to a particular infectious agent or disease. Examples of preferred epitopes include peptide epitopes of the influenza nucleoprotein (NP) gene, the tat, rev, gag and nef components of HIV, and epitopes from the E6 and E7 proteins of HPV. Additional preferred epitopes are those which are found to induce tolerance, such as the collagen involved in Rheumatoid arthritis.
More than one epitope may be encoded by the gene in order to increase the likelihood of an immune response. This is particularly important where the key epitopes which give rise to a protective immune responses have not been identified. Thus, in a further aspect of the present invention, the nucleic acid encodes more than one epitope capable of eliciting an immune response to a particular infectious agent or disease. This is particularly important where the key epitope(s) which give rise to a protective immune response have not been identified. Preferably, the epitope is present in a polypeptide (i.e., a polypeptide antigen) and therefore the polypeptide is encoded in a construct. Preferably, the construct is a multi-gene construct in which the antigens are coordinately expressed.
In a further embodiment of the present invention, where an immune response is desired to an infectious agent, all of the open reading frames from the pathogen""s genome are arranged together in a construct in order to form the coding region of the delivery vehicle. This has the advantage that all the antigens will be present and thus the likelihood of an immune response increased.
Basically, an expression-library is made of the pathogen DNA and the expression-library delivered in the delivery vehicle to the cell in order to elicit an immune response.
In a further embodiment of the present invention, DNA sequences encoding antigens specific for different diseases or infectious agents may be arranged together in a construct for expression in the delivery vehicle of the present invention. The antigens may be whole proteins or multiple or single epitopes thereof arranged as a single gene or as a multi-gene construct. The delivery means will therefore provide immune protection against a number of diseases specified by the antigens encoded within the DNA construct.
In a further embodiment of the present invention the delivery vehicle may comprise mixture of antigenic peptide/protein components. These may be derived from the same antigen, they may be different antigens from the same infectious agent or disease, or they may be from different infectious agents or diseases. The complex or mixture will therefore raise an immune response against a number of antigens and possibly a number of infectious agents or diseases as specified by the antigenic peptide/protein components of the delivery system.
In a further embodiment of the present invention the antigenic components of the complex or mixture may, or may not, be glycosylated depending on the requirement for glycosylation for the generation of a suitable immune response against the epitope or antigen.
In a further embodiment of the present invention, the DNA encoded antigen or the antigenic component of the complex or mixture may not necessarily encode the most immunodominant epitopes of the antigen. They may be epitopes which are more highly conserved between different strains of an infectious agent or disease, or may be epitopes which have limited, or no, mutational variation. Antigens may also be encoded as a series of subgenes which cover the whole antigen but the whole antigen may not necessarily be encoded in a single gene.
In a further embodiment of the present invention the DNA may encode additional factors which will have the effect of upregulating the immune response to the encoded antigen, or protein/peptide component of the delivery system. The additional factors may include cytokines for the general upregulation of specific components of the immune response e.g. interferon gamma, IL-2, IL-4, IL-10, IL-12, and GM-CSF; lymphokines; or co-stimulatory molecules such as B7-1, B7-2, ICAM-1 and ICAM-3. Alternatively, each factor may be included in a mixture of complex according to the invention in its polypeptide form, in that it may be co-administered with a nucleic acid and antigen according to the invention or it may be conjugated to the antigen or to a nucleic acid binding peptide so as to form part of the delivery complex.
The invention encompasses the delivery of a vector or nucleic acid to a cell (i.e., the use of a delivery vehicle).
As used herein, the phrase xe2x80x9cmeans for deliveringxe2x80x9d a vector to a cellxe2x80x9d or xe2x80x9cadapted for deliveryxe2x80x9d to a cell refers to any means, including viral and non-viral delivery means, by which it is possible to deliver nucleic acid and an antigenic peptide or protein associated with nucleic acid to a mammalian cell, including DNA/polycation complexes, self assembling virus like particles, viral vectors which are capable of delivering nucleic acid to a mammalian cell such as adenoviruses, retroviruses and adeno-associated viruses, microspheres which are used for delivery of DNA or protein to cells, e.g., polylactide glycolide polymers, and liposomes. Delivery means useful according to the present invention are well known to those skilled in the art and are described further herein.
Particularly preferred delivery vehicles are those which include polycation-condensed nucleic acid which in addition may be coupled with a ligand for targeting cells specifically or non-specifically.
The delivery vehicle may therefore include a nucleic acid condensing peptide, e.g., a polycationic heteropeptide, which binds DNA (i.e., a DNA binding peptide).
In another embodiment of the invention, the complex or mixture may include a peptide which does not necessarily participate in delivery of the vector or nucleic acid to the cell, but which is antigenic and thus serves to induce an immune response in a mammal upon entry of the delivery vehicle into the cell. The antigenic peptide may be a portion of a two-domain peptide comprising a DNA-binding amino acid sequence and the antigenic peptide. The two domains may be fused via a peptide bond to form a fusion polypeptide, wherein the antigenic domain is amino terminal and the DNA binding domain is carboxy terminal, or wherein the DNA binding domain is amino terminal and the DNA binding domain is carboxy terminal. Where a protein contains a DNA binding domain and also an epitope(s) to which an immune response is desired, the protein may be considered to be essentially equivalent to a two domain peptide as described herein.
Alternatively, the two domains may be separated by a cleavable linker such as a acid labile linkage or a peptide sequence cleavable by an endosomal protease such as cathepsin.
In another embodiment, it is also contemplated that the epitope is not part of a fusion protein but is directly absorbed onto a complex comprising a nucleic acid encoding an epitope in association with a DNA binding domain by electrostatic, hydrophobic, covalent or other interactions.
Where a DNA binding peptide is used in a complex or mixture of the invention, whether it be as part of a two domain fusion polypeptide or noncovalently associated with an epitope, it is envisioned that the DNA binding peptide binds the nucleic acid of the complex or mixture in a condensing reaction and therefore permits intimate association of the epitope portion of the complex or mixture with the nucleic acid.
In a further embodiment of the present invention the antigen encoding nucleic acid sequence may contain a signal sequence for secretion of the antigen outside the cell. Signal sequences useful in the present invention are well known to those skilled in the art and are, for example, described in Blobel and Dobberstein (1975), J. Cell Biol., 62, 852-862. The secreted antigen will be taken up by the secreting cell and other neighbouring cells, processed as an exogenous antigen, and presented in association with Class II MHC. The delivery vehicle of the present invention may contain a mixture of nucleic acids encoding an antigen, some with, and some without, the secretion signal sequence. Thus secreted and non-secreted antigen can be produced in the same cell and both Class I and Class II MHC presentation can be produced after a single transfection event.
An antigenic peptide useful as an antigen component of a delivery vehicle or in a two-domain peptide may be from the same or a different organism against which a DNA-based immune response is desired. An example of a preferred antigenic peptide for this purpose is the immunodominant peptide epitope(s) of influenza nucleoprotein (NP).
Upon entry of this delivery vehicle into a cell, an immune response will be elicited to the antigenic peptide. The immune response will be subsequently sustained and expanded by the expression of the antigenic protein encoding sequences also present in the delivery vehicle.
It is further preferred that the vector containing a nucleic acid encoding an antigen is maintained at a high copy number in dividing and non-dividing cells of a patient.
Thus, particularly preferred vectors useful according to the invention contain a sequence which permits maintenance of the vector in an episomal form. Such vectors may comprise a minimal origin of replication of a papilloma virus, a minichromosomal maintenance element of a papilloma virus, and a cloning site for inserting a nucleic acid encoding one or more antigens. This may be achieved by employing the BPV-I vector system comprising a plasmid harboring the BPV-I origin of replication, or a minimal origin plus minichromosomal maintenance element, as disclosed hereinbelow, and optionally the BPVxe2x80x94L E1 and E2 genes.
If desired, the antigenicity of selected proteins (e.g., targeting ligands) or peptides of the delivery vehicle, or other proteins encoded by the vector, (e.g., the BPV-1 E1 and E2 proteins) may be eliminated by introducing into their open reading frames sequences from the Epstein bar virus EBNA-1 protein encoding a Gly-Ala repeat, that suppresses antigen presentation of amino acid sequence linked in cis.
A composition of the present invention may be used in ex vivo gene therapy or in in vivo gene therapy in order to provide immune protection against various infectious agents and diseases.